A new, robust and reliable methodology for three-protease screening in a single-enzyme mode has been developed and verified, employing a multi-purpose peptide probe with three selectively cleavable sites furnished with four fluorophores. A triple-FRET-based single-excitation quadruple-emission concept for unambiguous sensing of trypsin, chymotrypsin and caspase-8 in the lowest detectable concentrations of 0.5 ng mL-1, 0.2 μg mL-1, and 2 U mL-1, respectively, has been applied and graphically depicted. Then the developed 4-dye probe has been also studied from the perspective of simultaneous two-protease screening, which was found only partially feasible, primarily due to unselective chymotrypsin cleavage.
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